Results: 4

1.
Fig. 2

Fig. 2. From: Differential Biological Activity of Disease-Associated JAK2 Mutants.

Biological effects of disease-associated JAK2 mutants. (A) Ba/F3-EpoR cells stably expressing GFP (vector), JAK2WT, JAK2V617F, JAK2K539L and JAK2T875N mutants were plated in triplicates in a 96-well plate in the absence of cytokine, and cell proliferation was measured using the WST assay. Asterisk indicates significant differences (p<0.01). Note that JAK2T875N-expressing cells exhibited significantly more cytokine-independent proliferation compared with cells expressing JAK2V617F and JAK2K539L. (B) Clonogenic growth of Ba/F3-EpoR cells expressing different JAK2 mutants was measured in the absence of cytokine in a methylcellulose-based colony assay. Results represent the mean ± SEM of three independent experiments. Asterisk indicates significant differences (p<0.005). Expression of JAK2T875N caused significantly more clonogenic growth of Ba/F3-EpoR cells than cells expressing JAK2V617F (p<0.005) or JAK2K539L (p<0.005).

Haiying Zou, et al. FEBS Lett. ;585(7):1007-1013.
2.
Fig. 1

Fig. 1. From: Differential Biological Activity of Disease-Associated JAK2 Mutants.

Catalytic activities of disease-associated JAK2 mutants. (A) Schematic structure of JAK2 and the locations of the disease-associated JAK2 mutants used in this study. (B) Mutant JAK2 proteins were immunoprecipitated and subjected to an in vitro kinase assay using a Stat5-derived peptide as a substrate. Phosphorylated peptides were measured by a scintillation counter (upper panel). Note that JAK2T875N mutant exhibited significantly more kinase activity than JAK2V617F (p<0.005) or JAK2K539L mutant (p<0.005), whereas JAK2V617F showed higher kinase activity than JAK2K539L (p<0.005). Immunoprecipitated samples were separated by SDS-PAGE and subjected to immuboblotting with anti-JAK2 antibody (bottom panel) to show equivalent expression of mutant JAK2 proteins.

Haiying Zou, et al. FEBS Lett. ;585(7):1007-1013.
3.
Fig. 3

Fig. 3. From: Differential Biological Activity of Disease-Associated JAK2 Mutants.

Transformation of erythroid progenitors by JAK2 mutants. BM from wild type C57/BL6 mice were transduced with equal titer retroviruses expressing GFP (vector), JAK2WT, JAK2V617F, JAK2K539L and JAK2T875N mutants and plated (1 × 104 cells per dish) in methycellulose medium in the absence or presence of Epo. CFU-E colonies were counted after 2 days. Results represent the mean ± SEM of three independent experiments. Note that JAK2V617F and JAK2K539L expression resulted in transformation of erythroid progenitors and gave rise to a large number of CFU-E in the absence or presence of Epo. However, JAK2T875N was less potent in transforming erythroid progenitors and gave rise to significantly less CFU-E in the absence or presence of Epo compared with JAK2V617F and JAK2K539L mutants (JAK2V617F vs JAK2K539L, p<0.05; JAK2V617F vs JAK2T875N, p<0.0005; JAK2K539L vs JAK2T875N, p<0.0005).

Haiying Zou, et al. FEBS Lett. ;585(7):1007-1013.
4.
Fig. 4

Fig. 4. From: Differential Biological Activity of Disease-Associated JAK2 Mutants.

Effects of JAK2 mutants on cell signaling. (A) Ba/F3-EpoR cells expressing different JAK2 mutants were starved in RPMI plus 0.5% BSA for 6 hrs. Cell lysates were prepared in RIPA buffer and subjected to immunoblotting with anti-phosphotyrosine (4G10) antibody. (B) Activation of the components of the JAK-Stat signaling pathway, which included JAK2, Stat5, Stat3, Pim-1 and Pim-2 were evaluated in Ba/F3-EpoR cells expressing JAK2 mutants by immunoblotting using phospho-specific antibodies followed by re-probing with total antibodies. (C) Effects of activating JAK2 mutants on other downstream signaling molecules, such as Shp2, Gab2, Akt and Erk1/2, were analyzed by immunoblotting using respective phospho-specific antibodies. Immunoblottings using antibodies against respective total proteins were used as loading controls. (D) Lysates from Ba/F3-EpoR cells expressing different JAK2 mutants were immunoprecipitated with anti-EpoR antibody followed by immunoblotting with anti-JAK2 antibody. The membrane was then re-probed with an antibody against EpoR. Cell lysates were also directly immunoblotted with anti-JAK2 antibody to confirm equivalent JAK2 expression in all mutant expressing cells. Note that association of EpoR with JAK2T875N was significantly less than with JAK2V617F or JAK2K539L.

Haiying Zou, et al. FEBS Lett. ;585(7):1007-1013.

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